Negative Chemical Ionization Mode Research Articles

Rapid determination of polybrominated diphenyl ethers (PBDEs) in fish using selective pressurized liquid extraction (SPLE) combined with automated online gel permeation chromatography-gas chromatography mass spectrometry (GPC-GC/MS)

A rapid method for the determination of seven predominant polybrominated diphenyl ethers (PBDEs) in fish was developed. The analysis could be completed in 1 h. The PBDEs in the sample were extracted with fat by selective pressurized liquid extraction (SPLE) using acid alumina as a fat retainer. After concentration, the extract was directly injected into online gel permeation chromatography with gas chromatography-mass spectrometry (GPC-GC/MS) in negative chemical ionization (NCI) mode. Method detection limits of seven PBDEs were between 0.002 and 0.005 ng g−1 fat. Quantitative recoveries ranging from 81.9% to 110.3% were obtained by analysis of spiked fish. The relative standard deviations were less than 15% (n = 6). The method was applied to a certified reference material of fish; all results were in the reference ranges. It was shown to be a rapid and reliable alternative in the routine analysis for PBDEs.

Analysis of pesticide residues by fast gas chromatography in combination with negative chemical ionization mass spectrometry

A combination of fast GC with narrow-bore column and bench top quadrupole mass spectrometer (MS) detector in negative chemical ionization (NCI) mode (with methane as reagent gas) is set up and utilized for the ultratrace analysis of 25 selected pesticides. The observed pesticides, belonging to the endocrine disrupting chemicals (EDCs), were from different chemical classes. A comparative study with electron impact (EI) ionization was also carried out (both techniques in selected ion monitoring (SIM) mode). The programmed temperature vaporizer (PTV) injector in solvent vent mode and narrow-bore column (15 m × 0.15 mm I.D. × 0.15 μm film of 5% diphenyl 95% dimethylsiloxane stationary phase) were used for effective and fast separation. Heptachlor (HPT) as internal standard (I.S.) was applied for the comparison of results obtained from absolute and normalized peak areas. Non-fatty food matrices were investigated. Fruit (apple – matrix-matched standards; orange, strawberry, plum – real samples) and vegetable (lettuce – real sample) extracts were prepared by a quick and effective QuEChERS sample preparation technique. Very good results were obtained for the characterization of fast GC–NCI-MS method analysing EDCs pesticides. Analyte response was linear from 0.01 to 150 μg kg −1 with the R 2 values in the range from 0.9936 to 1.0000 (calculated from absolute peak areas) and from 0.9956 to 1.0000 (calculated from peak areas normalized to HPT). Instrument limits of detection (LODs) and quantification (LOQs) were found at pg mL −1 level and for the majority of analytes were up to three orders of magnitude lower for NCI compared to EI mode. In both ionization modes, repeatability of measurements expressed as relative standard deviation (RSDs) was less than 10% which is in very good agreement with the criterion of European Union.

New Approaches for Determination of Glyphosate and Aminomethylphosphonic Acid from Different Tea SamplesProspects and Limits of Cleanup with Molecularly Imprinted Polymer and Titanium Dioxide

Glyphosate is the most commonly used herbicide worldwide. To identify glyphosate and its metabolite, aminomethylphosphonic acid (AMPA), in the difficult-to-analyze matrix of tea, new methods were developed. The main emphasis was placed on the cleanup procedure. Two different solid-phase extraction methods were tested and evaluated: one with molecularly imprinted polymers, and the other with immobilized titanium dioxide. The optimization was carried out on the basis of aqueous standard solutions and spiked tea extracts. Validated results were presented for pipet tips that contained immobilized titanium dioxide. After the extraction of glyphosate from tea samples using hydrochloric acid (0.1%), glyphosate and AMPA were concentrated on the adsorbent by pipetting up and down several times without changing the tip. The elution was carried out subsequent to a washing step with 5% ammonia. The extract was derivatized with 2,2,2-trifluoroacetic acid and 2,2,2-trifluoroethanol and then analyzed by GC with MS detection in the negative chemical ionization mode. Quantification was carried out in the selected-ion monitoring mode based on the m/z ratio of characteristic ions due to the ionization process. For glyphosate m/z = 370 was applied, and m/z = 351 for AMPA. The detection limit was 0.03 mg/kg tea for glyphosate and 0.006 mg/kg tea for AMPA. The recoveries for the tested working range (0.1 to 2.8 mg/kg tea) were low but constant at 14 and 12%, respectively. Altogether, the method presented here can be carried out easily and quickly, and it gives reliable information on the presence of the analytes in a sample. Quantification is also possible using standard addition.

Analysis of whole congener mixtures of polybromodiphenyl ethers by gas chromatography–mass spectrometry in both environmental and biological samples at femtogram levels

A sensitive and selective method for the determination of the whole congener distribution of polybromodiphenyl ethers in environmental and biological samples in one single instrumental run is described. The method is based on gas chromatography coupled to low-resolution mass spectrometry in negative ion chemical ionization mode. It allows determination of these compounds at concentration levels lower than 10 −14 g. A programmed temperature vaporization injector has been used to ensure maximum compound transfer to the chromatographic column while maintaining low thermal degradation levels of the more brominated congeners. Selectivity was increased by modification of the MS source parameters for optimization of the abundance of the high mass fragment ions. Under optimized condition, good repeatability (1.7–9.1%) and reproducibility (4.1–20%), and low detection limits, ranging between 1.5 and 15 pg ml −1, were obtained. These features afforded reliable quantification of these compounds in snow and human samples at the concentrations in which these compounds are found.

Ultrasensitive method for the determination of 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts by gas chromatography–high resolution mass spectrometry in mucosal biopsies of the lower esophagus

4-Hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts are formed by metabolic activation of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). NNK and NNN are considered carcinogenic to humans by the International Agency for Research on Cancer. Existing analytical methods for determination of HPB-releasing DNA adducts require 0.3-2.0 g of human target tissues such as lung and esophagus. For adduct determination in milligram amounts of biopsy samples, an ultrasensitive and specific method is presented using capillary gas chromatography coupled to a high-resolution mass spectrometer operated in the negative chemical ionization mode (GC-NCI-HRMS). The method has a limit of detection of 4.6 fmol HPB, a limit of quantification of 14.9 fmol HBP and a recovery of 45 +/- 15%. Intra- and inter-day imprecision for N = 6 samples were calculated with coefficients of variation of <3.1%. Method applicability was evaluated with biopsies of esophageal mucosa (N = 14) yielding 5.6 +/- 1.9 mg tissue and a mean adduct level of 6.13 +/- 9.35 pmol HPB/mg DNA.

Quantitative Determination of Ethyl Glucuronide in Sweat

In this study, ethyl glucuronide (EtG), a specific metabolite of ethanol, was for the first time detected in sweat after alcohol consumption by human volunteers. Sweat was collected using a sweat patch (PharmChek). After collection, chemicals accumulated on the patch were extracted with water and extracts were purified by solid phase extraction. EtG was determined by gas chromatography with mass spectrometric detection in negative chemical ionization mode. In parallel, the amount of sodium deposited on the patch was determined by capillary electrophoresis and used as a correction factor to calculate the volume of sweat accumulated on the patch and, hence, the concentration of EtG in sweat. The EtG sweat concentration observed ranged from 1.7 to 103.0 microg/L for alcohol consumption from 38.0 to 154.6 g equivalent pure ethanol. No EtG was detected in subjects who did not consume alcohol. Our results demonstrate that after ethanol consumption, EtG is detectable in sweat collected using a sweat patch. The simultaneous determination of sodium allows the estimation of the volume of sweat accumulated on the patch and to calculate the concentration of EtG in sweat. This represents the first quantitative determination of a xenobiotic in sweat collected using a sweat patch. This study suggests that EtG determination in sweat could represent an interesting alternative to urine or serum analysis for the control of abstinence of patients included in treatment programs.

Application of Pentafluorobenzyl and Hexafluoroisopropyl Esters for Retention Indexes in GC-Negative Ion Chemical Ionization MS

In order to use the linear temperature programmed retention index (LTRI) for the negative ion chemical ionization (NCI) mode in which n-alkanes cannot be detected, LTRIs were newly defined using homologous series of pentafluorobenzyl and hexafluoroisopropyl esters. The method for converting LTRI based on n-alkanes to the new LTRIs was also developed and applied to PCB and chlorinated pesticide analysis. The results demonstrated that the LTRI databases, which were developed based on n-alkanes, could be utilized for NCI analysis.

Quantification of Pentafluorobenzyl Oxime Derivatives of Long Chain Aldehydes by GC–MS Analysis

Negative ion mass spectrometric techniques, for compounds having good ionization properties, such as pentafluorobenzyl derivatives, are believed to be more sensitive than positive ion methods. Preparation of PFB oximes of fatty aldehydes from crude lipid extracts is problematic due to the release of free aldehydes from plasmalogens during derivatization. Accordingly, in these studies plasmalogens were removed by silicic acid column chromatography prior to pentafluorobenzyl derivatization. This simple purification step to remove plasmalogens is shown to facilitate the quantification of long-chain aldehydes by analysis of their pentafluorobenzyl oxime derivatives utilizing gas chromatography-mass spectrometry in the negative ion chemical ionization mode. The limit of detection for long chain fatty aldehydes using this method is 0.5 pmol and it is linear over two orders of magnitude. Silicic acid column chromatography followed by electrospray ionization mass spectrometry demonstrated that plasmalogens were removed (the detection limit for this analyses was <or=0.3 pmol). Furthermore, we have exploited the utility and sensitivity of this method to identify increases in hexadecanal and octadecanal in 3-amino-1,2,4-triazole treated human neutrophils.

Simultaneous determination of aliphatic and aromatic amines in ambient air and airborne particulate matters by gas chromatography-mass spectrometry

A gas chromatography-mass spectrometry (GC-MS) method has been proposed for the simultaneous determination of aliphatic and aromatic amines in ambient air and airborne particulate matters (PMs). The method includes collection of the particulate matters (PM2.5 and PM10) using dichotomous Partisol 2025 sampler followed by extraction of the compounds into acidic solution, and pre-concentration of the compounds by percolating the air samples through the acidic solution, then ion-pair extraction of amines with bis-2-ethylhexylphosphate and derivatisation with isobutyl chloroformate prior to their GC-MS analysis in both electron impact and positive and negative ion chemical ionisation mode as their isobutyloxycarbonyl (isoBOC) derivatives. In the present study, ambient air and airborne particulate samples collected in Zonguldak province during summer and winter times of 2006–2007 were analysed for aliphatic and aromatic amines by the proposed method and the method was shown to be suitable for the simultaneous determination of these compounds at the levels of pgm−3 in air and airborne particulate samples. The seasonal distributions of bioactive amines in concentrations in ambient air and airborne PMs were evaluated as they are significant for the estimation of their effects on the environment and human health. The concentration levels of water soluble amines fluctuate significantly within a year with higher means and peak concentrations, probably due to the increased emissions from coal-fired domestic and central heating, in the winter times compared to the summer times. The results indicated that the relative amine content in particulates modulates with molecular mass and time of the year and the relative amine content especially in fine fractions of inhalable airborne particulates increases with the molecular mass of species but decreases with temperature.

Determination of PBDEs in human breast adipose tissues by gas chromatography coupled with triple quadrupole mass spectrometry

The potential of gas chromatography/tandem mass spectrometry with a triple quadrupole analyzer for determination of 12 polybrominated diphenyl ethers in human breast tissues has been investigated. After extraction with hexane, two purification procedures-automated normal-phase high-performance liquid chromatography and solid-phase extraction-were assayed. Both electron impact ionization, in selected reaction monitoring mode, and negative chemical ionization, in selected ion recording mode, were tested for the optimum determination of analytes. Isotopically labeled standards were added before extraction as surrogates: [13C]BDE47, [13C]BDE99 and [13C]BDE153 for electron impact ionization, and p,p'-DDE-d (8) for negative chemical ionization. The method was validated in terms of accuracy, precision, limits of detection and limits of quantification, using human breast tissue spiked at three levels in the range 1-50 ng/g (5-250 ng/g for BDE209). The analytical approach using solid-phase extraction cleanup followed by gas chromatography/mass spectrometry (negative chemical ionization ) led to lower detection limits (0.006-2 ng/g) and allowed the determination of the most problematic congener, BDE209, whose poor sensitivity made difficult its determination at low residue levels. Special attention was given to the confirmation of the compounds detected in samples in order to avoid reporting false positives. Two tandem mass spectrometry transitions or three m/z ions were selected for each analyte when using electron impact ionization or negative chemical ionization modes, respectively. In both cases, the transition to ion intensity ratio was used as a confirmation parameter. The method developed was applied to the analysis of real human samples. Several brominated diphenyl ethers (congeners 47, 100, 99, 154, 153, 183 and 209) were detected in the range 0.08-0.23 ng/g.

Haemoglobin adducts as biomarkers of exposure to tobacco-related nitrosamines

A sensitive gas chromatography/mass spectrometry (GC/MS) method was developed to measure nitrosamine–haemoglobin adducts (HPB-Hb) (4-hydroxy-3-pyridinyl-1-butanone) at trace levels in red blood cells of smoking and non-smoking mothers and their newborn babies. GC/MS methods with chemical ionization (CI) of methane reagent gas in both positive and negative ion mode as well as electron ionization (EI) were studied to determine differences in sensitivity among the various ionization methods. Detection limits using both positive and negative chemical ionization modes were found to be 30 fmol HPB, whereas detection using electron impact modes yielded a detection limit of 80 fmol HBP. In order to apply the various methods of detection to tobacco-exposed samples from human populations, we characterized adduct levels in maternal as well as paired fetal samples obtained from mothers exposed to tobacco smoke during pregnancy. Maternal samples were characterized using serum cotinine levels and were classified as non-smokers, passively smoke-exposed women, less than one pack per day smokers and greater than one pack per day smokers. Paired maternal and fetal blood samples were obtained at delivery for qualitative and qualitative analysis of nitrosamine adducts. Comparative derivatization of HPB released under alkaline hydrolysis conditions was performed using O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) and 2,3,4,5,6-pentafluorobenzoylchloride (PFBC). Both negative CI and positive CI modes of analysis were compared to the more widely accepted EI modes of mass spectrometric analysis. These results suggest that both NICI and PICI modes of detection offer a greater sensitivity of adduct characterization when compared with EI ionization techniques and that either NICI or PICI modes are preferably applicable towards the detection of human biomarker assessment of tobacco-related nitrosamines.

Determination of aromatic amines in hair dye and henna samples by ion-pair extraction and gas chromatography–mass spectrometry

A gas chromatography–mass spectrometry (GC–MS) method has been proposed for the determination of carcinogenic and toxic aromatic amines in hair dye, henna and dyed hair samples. The method includes ion-pair extraction of aromatic amines from aqueous samples with bis-2-ethylhexylphosphate (BEHPA) released after solving the samples in acidic solution followed by sonication, derivatisation of compounds with isobutyl chloroformate (IBCF) and their GC–MS analysis in both electron impact (EI) and positive and negative ion chemical ionisation (PNICI) mode as their isobutyloxycarbonyl ( isoBOC) derivatives. The obtained recoveries of aromatic amines ranged from 92.2 to 98.4% and the precision of this method, as indicated by the relative standard deviations (RSDs) was within the range of 0.7–4.2%. The detection limits obtained from calculations by using GC–MS results based on signal-to-noise ratio (S/N)= 3 were within the range from 0.02 to 0.20 ng/g. In the present study, the commercially available 54 permanent hair dye, 35 modified or natural henna and 15 dyed hair samples were analysed for the aromatic amines by the proposed method and the method was shown to be suitable to determine the aromatic amine ingredients and metabolites of these commercial products.

Detection of Dechlorane Plus in Residential Indoor Dust in the City of Ottawa, Canada

Dechlorane plus (DP), a flame retardant recently identified in the air and sediment of the Great Lakes region, has been detected in residential indoor dust in the city of Ottawa, Canada using gas chromatography-mass spectrometry under negative chemical ionization mode. DP was detected in all dust samples with a concentration range of 2.3 to 182 ng g(-1), except for one dust sample (5683 ng g(-1)) which was much higher. The levels of DP in indoor dust were several times higher than those in the sediment of the Great Lakes. The presence of DP in indoor environments could have implications for potential human exposure to this chemical. The daily human intake of DP from dust was estimated using dust ingestion data from the U.S. Environmental Protection Agency. The large variation of the ratio of syn- and anti-DP isomers in the dust samples also suggests a complex degradation process of DP in the environment.

Cross validation of multiple methods for measuring pyrethroid and pyrethrum insecticide metabolites in human urine

The objective of our study was to compare three vastly different analytical methods for measuring urinary metabolites of pyrethroid and pyrethrum insecticides to determine whether they could produce comparable data and to determine if similar analytical characteristics of the methods could be obtained by a secondary laboratory. This study was conducted as a part of a series of validation studies undertaken by the German Research Foundation's Committee on the Standardization of Analytical Methods for Occupational and Environmental Medicine. We compared methods using different sample preparation methods (liquid-liquid extraction and solid-phase extraction with and without chemical derivatization) and different analytical detection methods (gas chromatography-mass spectrometry (single quadrupole), gas chromatography-high resolution mass spectrometry (magnetic sector) in both electron impact ionization and negative chemical ionization modes, and high-performance liquid chromatography-tandem mass spectrometry (triple quadrupole) with electrospray ionization). Our cross validation proved that similar analytical characteristics could be obtained with any combination of sample preparation/analytical detection method and that all methods produced comparable analytical results on unknown urine samples.

Quantification of Lorazepam and Lormetazepam in Human Breast Milk Using GC-MS in the Negative Chemical Ionization Mode

Lormetazepam (Loramet is a benzodiazepine mainly used as an hypnotic to treat insomnia. Lorazepam (Temesta) is used as an anxiolytic, tranquilizer, sedative, and anticonvulsant, and it is the major metabolite of lormetazepam. In this study, we designed a method to simultaneously detect and quantify these substances in human breast milk. Solid-phase extraction of 2 mL of milk was followed by derivatization with a trimethylsilyl reagent. Separation and detection was performed using gas chromatography coupled to mass spectrometry in the negative chemical ionization mode. Calibration curves were linear in the ranges of 10-200 and 1-20 ng/mL for lorazepam and lormetazepam, respectively. Limits of detection were estimated at 0.016 ng/mL for lormetazepam and 0.100 ng/mL for lorazepam. Our method was applied to real case samples from a woman receiving both benzodiazepines. Lorazepam concentrations varied from 55.3 to 123.1 ng/mL, and lormetazepam concentrations varied from 1.7 to 7.3 ng/mL.

Synthesis and characterization of deoxynivalenol glucuronide: Its comparative immunotoxicity with deoxynivalenol

Deoxynivalenol (DON) is a mycotoxin commonly contaminating wheat, barley and corn. DON glucuronide (DONGLU) is a major DON metabolite. We synthesized and purified DONGLU and tested its immunotoxicity, hypothesizing that DONGLU would be much less toxic to K562 cells compared with DON. DONGLU was synthesized using rat liver microsomes, uridine-5′-diphosphoglucuronic acid and DON, and purified with a Sephadex LH-20 column and reverse phase HPLC. β-Glucuronidase hydrolysis formed a product with retention time and UV spectrum identical with DON. Using atmospheric pressure chemical ionization in negative mode, the molecular mass (M−1) of purified DONGLU was 471 g/mol; in agreement with an expected molecular weight of 472 g/mol. MS and NMR indicated that the glucuronide moiety was conjugated with the carbon-3-hydroxyl group of DON. The cytotoxicity of DON and DONGLU were compared in cell culture using human erythroleukemia cell line K562. Fifty percent inhibition of cell number was observed with a DON concentration of 1.31 μM using a methylthaizol tetrazolium (MTS) cell viability assay whereas no significant cytotoxicity was observed for DONGLU at up to 270 μM. DONGLU did not influence DON toxicity at 0.5 μM, 1.3 μM and 8.4 μM concentration combinations of each compound. These data verified that DONGLU is a detoxification product of DON.

Microwave-assisted extraction (MAE) for the determination of polybrominated diphenylethers (PBDEs) in sewage sludge

An efficient microwave-assisted extraction (MAE) method has been developed and evaluated for the quantification of eight major polybrominated diphenylethers (PBDEs) in sewage sludge. The PBDEs were extracted from wet and dry sludge in a microwave extraction unit using a hexane/acetone mixture for 35 min at a controlled temperature of 130 degrees C. The extract was concentrated, cleaned up on a silica gel column, and analyzed by gas chromatography/mass spectrometry (GC/MS) in the negative chemical ionization (NCI) mode. The MAE procedure exhibited higher extraction efficiency, specifically for BDE (brominated diphenylether) 209, than the conventional Soxhlet extraction. The test congeners were clearly separated under specific instrumental operating conditions, at a source temperature of 230 degrees C and a column length of 20 m. The present analytical method showed recovery efficiencies ranging from 80 to 110% when applied to the PBDE-free sludge spiked with eight PBDE congeners. The efficiency of the MAE method was confirmed using sludge obtained from four sewage treatment plants (STPs). The results indicate that BDE 47, 99, and 209 are the most abundant congeners present in these sewage sludges, which is consistent with previous reports.

Time-resolved hair analysis of MDMA enantiomers by GC/MS-NCI

The aim of the study was to determine the enantioselective disposition of 3,4-methylenedioxymethamphetamine (MDMA) and other amphetamine-type stimulants (ATS) in segmented hair specimens of self-declared ecstasy abusers, who took part in a double-blind placebo-controlled six-way crossover study during approximately 7 weeks, during which they received a 75 and a 100 mg dose of racemic MDMA twice. Hair specimens were washed and cut into pieces of 2 cm length. After digestion and solid phase extraction, the enantiomers were derivatized with a chiral agent (2 S,4 R)- N-heptafluorobutyryl-4-heptafluorobutoyloxy-prolyl chloride, developed at the authors laboratory and quantified by gas chromatography coupled to mass spectrometry operating in the negative chemical ionization mode. Most of the hair specimens that were tested positive for MDMA showed a predominance of the ( R)-enantiomer. The R/ S ratios of MDMA varied between 1.02 and 2.75 and total concentrations ranged from 0.1 to 20.1 ng/mg. The enantiomers of its metabolite 3,4-methylenedioxyamphetamine (MDA) were also quantified in most hair segments. The R/ S ratios of MDA varied between 0.60 and 1.60, while the concentrations of the enantiomers ranged from 10 to 160 pg/mg hair. When segmental analysis was performed on single hair specimens, no inversion of the R versus S ratios of MDMA and MDA was observed. The predominance of ( R)-MDMA in hair was in accordance with those already published for other matrices. Furthermore, both enantiomers of amphetamine (AM) were also detected in hair segments of four volunteers and the R/ S ratios ranged from 1.00 to 1.47.

A Microwave-assisted Extraction Method for the Determination of Synthetic Musks in Sewage Sludge

Abstract An efficient microwave-assisted extraction (MAE) procedure has been optimized for the determination of polycyclic and nitro musks in sewage sludge. A centrifuged sludge cake sample (from 1 to 2.5 g ), mixed with 10 g of anhydrous sodium sulfate in a Teflon cell, was extracted with 30 mL of a 1:1 (v/v) mixture of acetone and hexane for 35 min, including a 15-min ramp with a 20-min hold at a controlled temperature of 110°C. After 30 min of cooling, the extract was evaporated and cleaned up on a silica gel column. A concentrated sample extract was then analyzed by gas chromatography/mass spectrometry (GC/MS) in the electron impact mode for the polycyclic musks and in negative ion chemical ionization mode for the nitro musks. This new method has been validated by comparing it to an established accelerated solvent extraction (ASE) method, with method detection limits ranging from 27 to 41 ng/g for the polycyclic musks, and approximately 4 ng/g for the nitro musks. Recoveries of most compounds in spiked samples ranged from 80 to 105%, although the MAE method produced lower recoveries for the nitro musks than the ASE method in archived sludge samples. The results generated by this MAE method were consistent with those prepared by previously reported techniques such as supercritical fluid extraction.

Mass spectrometric analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts in human lung

An improved analytical method was developed for the analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts in lung samples of patients undergoing surgery for lung cancer. HPB-releasing adducts can be formed by metabolic activation of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N′ -nitrosonornicotine, and have been reported to play an important role in tobacco carcinogenesis. [2,2,3,3-D 4]HPB (D 4-HPB) was used as an internal standard, and HPB released by acid hydrolysis of DNA was determined by gas chromatography/mass spectrometry in the negative ion chemical ionisation mode. The method is sensitive with a limit of detection of 5.9 fmol HPB and a limit of quantification of 15.2 fmol HBP/mg DNA. The recovery of HPB was 82 ± 17% and the background response was 10.1 ± 1.8 fmol HPB/sample. The concentration of HPB-releasing lung DNA adducts was significantly higher ( p < 0.0001) in 21 self-reported smokers compared to in 11 self-reported nonsmokers (404 ± 258 fmol versus 59 ± 56 fmol HPB/mg DNA, respectively). HPB-releasing hemoglobin adduct concentrations were only marginally higher in a subset of 12 smokers compared to in 7 nonsmokers (63 ± 53 fmol versus 42 ± 34 fmol HPB/g hemoglobin; p = 0.36). No correlation was found between HPB-releasing adducts in DNA and hemoglobin ( p = 0.074).